Genomic and transcriptomic profiling reveals key molecules in metastatic potentials and organ-tropisms of hepatocellular carcinoma.

Metastasis is a landmark event for rapid postsurgical relapse and death of HCC patients. Although distinct genomic and transcriptomic profiling of HCC metastasis had been reported previously, the causal relationships of somatic mutants, mRNA levels and metastatic potentials were difficult to be established in clinic. Therefore, 11 human HCC cell lines and 7 monoclonal derivatives with definite metastatic potentials and tropisms were subjected to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). TP53, MYO5A, ROS1 and ARID2 were the prominent mutants of metastatic drivers in HCC cells. During HCC clonal evaluation, TP53, MYO5A and ROS1 mutations occurred in the early stage, EXT2 and NIN in the late stage. NF1 mutant was unique in lung tropistic cell lines, RNF126 mutant in lymphatic tropistic ones. PER1, LMO2, GAS7, NR4A3 expression levels were positively associated with relapse-free survival (RFS) of HCC patients. The integrative analysis revealed 58 genes exhibited both somatic mutation and dysregulated mRNA levels in high metastatic cells. Altogether, metastatic drivers could accumulate gradually at different stages during HCC progression, some drivers might modulate HCC metastatic potentials and the others regulate metastatic tropisms.

miR-296-5p suppresses stem cell potency of hepatocellular carcinoma cells via regulating Brg1/Sall4 axis.

Epithelial-mesenchymal transition (EMT), a pivotal event during cancer progression such as relapse and metastasis, is positively correlated with the stemness potency of tumor cells. Our previous study showed that miR-296-5p attenuated EMT program of hepatocellular carcinoma cells (HCC) through NRG1/ERBB2/ERBB3 signaling. In the present study, we uncovered that miR-296-5p was able to inhibit the stemness potency of HCC by decreasing the number and size of tumorspheres, downregulating the expression of CSC biomarkers and hampering the ability of tumorigenesis in NOD/SCID mice. Brahma-related gene-1 (Brg1), as the target protein of miR-296-5p detected by bioinformatics methods, activates a series of downstream cascades through directly binding to Sall4 promoter and enhancing Sall4 transcription. Importantly, the higher expressions of Brg1 and Sall4 in tumor tissues of HCC patients suggest poorer prognoses after surgical extraction. In conclusion, miR-296-5p exerts an inhibitory effect on stemness potency of HCC cells via Brg1/Sall4 axis.

Antimetastatic Effect of Fucoidan-Sargassum against Liver Cancer Cell Invadopodia Formation via Targeting Integrin αVß3 and Mediating αVß3/Src/E2F1 Signaling.

Background: Fucoidan is a fucose-enriched, sulfated polysaccharide found in brown algae; in recent years, this polysaccharide has been found to exert several biological effects, including antitumor effects, such as antiproliferation, activating apoptosis, and anti-angiogenesis of cancer cells. However, the antimetastatic effect of fucoidan and the related targeting receptors remain unknown. In the present study, we examined the inhibition of invadopodia formation and underlying mechanism of fucoidan on human liver cancer cells. Methods: We used 98% purified fucoidan from Sargassum species to treat the hepatocellular carcinoma (HCC) cells SMMC-7721, Huh7 and HCCLM3 in vitro and the HCCLM3 cell line in vivo. The HCC cells were cultured with various concentrations of Fucoidan-Sargassum (0-30 mg/mL). Migration, invasion and wound healing assays were performed to determine the antimetastatic effect of fucoidan on the HCC cells. Western blot analysis and immunofluorescence staining were conducted to determine the expression levels of invadopodia formation-regulating proteins and the targeting membrane receptor proteins. Results: Fucoidan-Sargassum inhibited the migration and invasion of HCC SMMC-7721, Huh7 and HCCLM3 cells in a dose-dependent manner. In the HCCLM3 cells, Fucoidan-Sargassum also decreased the expression levels of invadopodia-related proteins including Src, Cortactin, N-WASP, ARP3, CDC42, MMP2, MT1-MMP, and the targeting receptors integrin αV and ß3 in a dose-dependent manner. Fucoidan-Sargassum also increased the levels of endoplasmic reticulum-related proteins, including GRP78, IRE1, SPARC, and the type IV collagen receptor proteins integrin α1 and ß1. In vivo, Fucoidan-Sargassum reduced the size of liver tumors and decreased the number of lung metastatic foci in nude mice with hepatocellular carcinoma xenografts. Conclusion: These findings indicate that Fucoidan-Sargassum has an antimetastatic effect on SMMC-7721, Huh7 and HCCLM3 liver cancer cells, and the underlying mechanism involves targeting ITGαVß3 and mediating the ITGαVß3/SRC/E2F1 signaling pathway. These results suggest that Fucoidan-Sargassum may be a promising therapeutic antimetastatic compound in the development of a metastasis-preventive drug for treating liver cancer.

miR-296-5p suppresses EMT of hepatocellular carcinoma via attenuating NRG1/ERBB2/ERBB3 signaling.

BACKGROUND: Accumulation of evidence indicates that miRNAs have crucial roles in the regulation of EMT-associated properties, such as proliferation, migration and invasion. However, the underlying molecular mechanisms are not entirely illustrated. Here, we investigated the role of miR-296-5p in hepatocellular carcinoma (HCC) progression. METHODS: In vitro cell morphology, proliferation, migration and invasion were compared between HCC cell lines with up- or down-regulation of miR-296-5p. Immunofluorescence and Western blot immunofluorescence assays were used to detect the expression of EMT markers. Bioinformatics programs, luciferase reporter assay and rescue experiments were used to validate the downstream targets of miR-296-5p. Xenograft nude mouse models were established to observe tumor growth and metastasis. Immunohistochemical assays were conducted to study the relationships between miR-296-5p expression and Neuregulin-1 (NRG1)/EMT markers in human HCC samples and mice. RESULTS: miR-296-5p was prominently downregulated in HCC tissues relative to adjacent normal liver tissues and associated with favorable prognosis. Overexpression of miR-296-5p inhibited EMT along with migration and invasion of HCC cells via suppressing NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling in vitro. More importantly, miR-296-5p disrupted intrahepatic and pulmonary metastasis in vivo. NRG1, as a direct target of miR-296-5p, mediates downstream biological responses. In HCC tissues from patients and mice, the levels of miR-296-5p and NRG1 also showed an inverse relationship. CONCLUSIONS: miR-296-5p inhibited EMT-related metastasis of HCC through NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling.

miR-106b-5p promotes stem cell-like properties of hepatocellular carcinoma cells by targeting PTEN via PI3K/Akt pathway.

BACKGROUND: The miRNA miR-106b-5p has been previously reported to be increased in hepatocellular carcinoma (HCC) tissues compared to cirrhotic tissues. The purpose of this study was to detect its expression in HCC cell lines with distinct metastatic potentials and to explore the molecular mechanisms underlying HCC stemness and migration. METHODS: miR-106b-5p expression was studied in HCC tissues and cell lines. In vitro cancer stem cell (CSC)-like properties, cell migration and invasion were compared between HCC cell lines with upregulation or downregulation of miR-106b-5p. In vivo tail vein injection models were established to evaluate the role of miR-106b-5p in lung metastasis. Bioinformatics programs, luciferase reporter assay and rescue experiments were used to validate the downstream targets of miR-106b-5p. The relationship between the expression of the targeted gene and clinicopathological parameters was also analyzed. RESULTS: miR-106b-5p expression was higher in HCC tissues and cell lines than that in non-tumor tissues and hepatocyte Chang liver, respectively. Upregulation of miR-106b-5p exhibited a promoting role in CSC properties, cell migration and activation of phosphatidylinositol-3 kinase (PI3K)/Akt signaling in vitro, as well as in lung metastasis in vivo. However, downregulation of miR-106b-5p exhibited the opposite effect. Furthermore, PTEN was verified as a direct target of miR-106b-5p. Upon clinicopathological analysis, lower level of PTEN was significantly associated with more aggressive characteristics. Patients with high PTEN expression had longer overall survival and disease-free survival. CONCLUSION: miR-106b-5p promotes HCC stemness maintenance and metastasis by targeting PTEN via PI3K/Akt pathway. Inhibition of miR-106b-5p might be effective therapeutic strategies to treat advanced HCC.

miR-612 suppresses stem cell-like property of hepatocellular carcinoma cells by modulating Sp1/Nanog signaling.

In our previous study we found that miR-612 negatively regulated stem cell-like property and tumor metastasis of hepatocellular carcinoma cells (HCC). In this study, we try to elucidate underlying mechanism of the regulation, and find that miR-612 inversely modulate the mRNA and protein level of epithelial cell adhesion molecule as well as CD133, negatively regulate the numbers and sizes of tumor spheres, directly inhibit the protein level of Sp1, and subsequently reduce transcription activity of Nanog. Of importance, the higher levels of Sp1 and Nanog in biopsies are the more unfavorable prognoses of HCC patients are found after tumor resection. Taken together, miR-612 has a suppressive role on HCC stemness via Sp1/Nanog signaling pathway.

[Expression of cyclooxygenase-2 in bladder transitional cell carcinoma and the significance thereof].

OBJECTIVE: To study the expression of cyclooxygenase-2 (COX-2) in bladder transitional cell carcinoma (Tcc) and the clinical significance thereof. METHODS: Immunohistochemistry was used to detect the expression of COX-2 in 56 specimens of bladder Tcc and 10 specimens of normal bladder tissue, all resected during operation. RESULTS: No expression of COX-2 was detected in the 10 specimens of normal bladder tissue and in the 56 specimens of bladder Tcc the positive rate of COX-2 was 55.36%. The expression of COX-2 in bladder Tcc was closely correlated with the grade and stage of tumor (both P < 0.01). CONCLUSION: COX-2 expression in bladder Tcc is closely correlated with the grade and stage.